Ninjinyoeito Prevents Onset of Depression-Like Behavior and Reduces Hippocampal iNOS Expression in Senescence-Accelerated Mouse Prone 8 Mice

Late-life depression is a globally prevalent disorder. Ninjinyoeito (NYT), a traditional Japanese herbal medicine, attenuates depressive symptoms in older patients. However, the mechanisms underlying the antidepressive effect of NYT are unknown. In this study, we investigated the mechanism of the action of NYT using senescence-accelerated mouse prone 8 (SAMP8) mice, which exhibit accelerated aging. SAMP8 mice were treated with NYT starting at 12 weeks of age. Twelve-week-old SAMP8 mice did not show prolonged immobility time in the tail suspension test compared with age-matched SAMR1 mice (normal aging control). At 34 weeks of age, vehicle-treated SAMP8 mice displayed prolonged immobility time compared with SAMR1 mice. NYT-treated SAMP8 mice showed a shorter immobility time than that of vehicle-treated SAMP8 mice. Notably, NYT decreased hippocampal inducible nitric oxide synthase (iNOS) expression in SAMP8 mice. There was no difference in iNOS expression between SAMR1 and vehicle-treated SAMP8 mice. Subchronic (5 days) administration of an iNOS inhibitor, 1400 W, shortened the immobility time in SAMP8 mice. These results suggest that NYT prevents an increase in immobility time of SAMP8 mice by decreasing iNOS levels in the hippocampus. Therefore, the antidepressive effect of NYT in older patients might be mediated, at least in part, by the downregulation of iNOS in the brain. Our data suggest that NYT is useful to prevent the onset of depression with aging.


Introduction
Depression in older adults is common, with 8.2%-41.8% of this population exhibiting depressive symptoms globally [1][2][3][4][5]. Severe depression, in particular, is associated with a marked reduction in the quality of life in older patients [6]. Furthermore, depression in older adults is suggested to increase the risk of frailty [7]. Terefore, therapy for depression is important for healthy living in this age group.
Ninjinyoeito (NYT) is a traditional Japanese herbal medicine (Kampo medicine) that originates in China. NYT is composed of 12 medicinal herbs (Table 1). Te extract of NYT is approved in Japan and is prescribed for symptoms such as fatigue, cold limbs, anorexia, night sweats, and anemia. Recent clinical studies have demonstrated that NYT has antidepressive efects in older patients with various diseases, including chronic obstructive pulmonary disease and Alzheimer's disease [8][9][10]. However, the mechanisms underlying the therapeutic efectiveness of NYT for depression in older patients are unclear.
A commonly used antidepressant, fuoxetine, inhibits hippocampal inducible nitric oxide synthase (iNOS) gene expression and alleviates depression-like behavior in mice exposed to chronic mild stress [16]. Te specifc iNOS inhibitor N- (3-(aminomethyl)benzyl)acetamidine (1400 W) also attenuates stress-induced depression-like behavior [17]. Tese fndings suggest that inactivation of iNOS is involved in the antidepressive-like efect of these drugs. Furthermore, aminoguanidine, an iNOS inhibitor, ameliorates depressionlike behavior and blocks the increase in hippocampal iNOS expression [18]. Intrahippocampal administration of aminoguanidine has an antidepressive-like efect in mice subjected to chronic mild stress [19]. Tus, hippocampal iNOS may play a key role in the antidepressive action of these drugs.
In the present study, we investigate whether NYT attenuates depression-like behavior and modulates hippocampal iNOS expression in SAMP8 mice. Furthermore, we examine the efect of an iNOS inhibitor on the depressionlike behavior in these mice, with the aim of clarifying the mechanisms underlying the antidepressive action of NYT in older patients.

Animals.
All procedures involving animal care and use were carried out in accordance with the regulations stipulated by the Experimental Animal Care and Use Committee of Fukuoka University (#1714148, #1915122). Male SAMP8 and SAMR1 mice, 10 weeks of age, were purchased from Japan SLC (Hamamatsu, Japan). Mice were housed individually in cages at a temperature of 23 ± 2°C and a relative humidity of 60 ± 10% under a 12/12 h light-dark cycle (lights on: 07: 00-19: 00). Food and water were available ad libitum. Body weight was measured weekly to monitor health status. Sample size was determined on the basis of reports examining the efects of Kampo medicines on depression-like behavior in mice [20,21]. Tis study is reported following the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Supplementary Table 1).

Drugs and Reagents.
NYT extracts (lot. nos. 372176700 and 352223500) were gifted by Tsumura & Co. (Tokyo, Japan). Te composition of the NYT extract and the details of its preparation have been previously described [22]. NYT was added to the bottled drinking water so that the mice ingested NYT at doses of 300 or 1,000 mg/kg/day (NYT300 or NYT1000) from 12 to 35 weeks of age. Bottled water was replaced weekly. Based on body weight and the amount of water that the mice drank over a week, the amount of NYT to be added to the new bottle the following week was calculated. Te iNOS inhibitor 1400 W (Selleck Chemicals, Houston, TX, USA) was dissolved in saline. For SAMP8 mice, 1400 W was administered intraperitoneally once a day for 5 days. Te ffth 1400 W administration was carried out 1 h before the tail suspension test.

Tail Suspension Test.
Te tail suspension test was carried out according to a previous report with modifcation [23]. Mice were suspended by the tail using rubber tape for 10 min. Duration of immobility was blindly measured throughout the 10 min period.

Elevated plus Maze Test.
Te procedure was based on that described by Egashira et al. [24]. Te plus maze apparatus was composed of two open and two closed arms elevated to a height of 50 cm. Te luminance on the open arm was about 45 lux. Te time spent in the open and closed arms and the number of closed arm entries were used for analyses. Te test duration was 5 min.
Behavioral tests were carried out at 34 weeks of age to investigate the efect of NYT, and brain samples were harvested 1 week later. Another experimental mouse group was subjected to the tail suspension test at 12 weeks of age. To analyze the efects of 1400 W, 37-51-week-old SAMP8 and SAMR1 mice were used.

Statistical Analysis.
Data were evaluated using one-way analysis of variance followed by Tukey's or Dunnett's multiple comparisons test using Prism 6.04 software (GraphPad, La Jolla, CA, USA). Tree outliers and one outlier were removed from the datasets in Figure1 (P8 + 1400 W, n = 3) and Figure 2(b) (P8 + NYT300, n = 1), respectively. Outliers were identifed using the outlier identifcation (ROUT) method, and moreover, removed if their value was more than 2.5-fold that of the group mean. One mouse dataset (P8 + water, n = 1) could not be analyzed in the elevated plus maze test because the video camera did not work properly. Te criterion for statistical signifcance was p < 0.05. Data are shown as mean ± standard error of the mean (SEM).

NYT Attenuates Depression-Like Behavior in SAMP8
Mice in the Tail Suspension Test. Immobility time in the tail suspension test was used to evaluate the efect of NYT on depression-like behavior. Tirty-four-week-old SAMP8 mice showed a prolonged immobility time compared with that of age-matched SAMR1 mice ( Figure 3). NYT1000 signifcantly shortened the immobility time in SAMP8 mice, while NYT300 had no efect. Another experiment showed immobility time of SAMP8 mice was comparable to that of SAMR1 mice at 12 weeks of age (Supplementary Figure 1), suggesting that SAMP8 mice do not show depression-like behavior at 12 weeks of age. Tese results suggest that NYT has an antidepression-like efect in mice with age-related depression. Te time spent in the closed arm of the elevated plus maze did not difer among the groups, nor did the time spent in the open arm (Figures 4(a) and 4(b)), suggesting that SAMP8 mice did not exhibit anxiety-like behavior in the elevated plus maze. SAMP8 mice demonstrated a tendency toward an increase in the number of entries into the closed arm (Figure 4(c)), but the observed tendency was not signifcant. NYT did not afect the behavior of SAMP8 mice in this test. Considering that the time spent in each arm and the number of entries into the closed arm were not signifcantly diferent between SAMP8 and SAMR1 mice, locomotion and anxiety levels of SAMP8 mice were similar to those of SAMR1 mice. NYT did not afect spontaneous locomotion and anxiety. Terefore, the antidepression-like efect of NYT may not be implicated in spontaneous locomotion.

NYT Decreases iNOS Expression in the Hippocampus.
Te efect of NYTon iNOS expression was examined because iNOS is implicated in depression-like behavior [17,18]. NYT1000 decreased iNOS levels in the hippocampus, but did not afect frontal cortical iNOS levels ( Figure 5). Tere were no diferences in iNOS levels between SAMP8 and SAMR1 mice in either tissue. Tese results suggest that NYTexerts its antidepression-like efect in SAMP8 mice, at least in part, by downregulating iNOS, although iNOS does not appear to be a direct cause of the depression-like behavior in these animals.  SAMP8 mice, supporting the hypothesis that NYTattenuates depression-like behavior by downregulating iNOS.

NYT Tends to Decrease Aryl Hydrocarbon Receptor (AhR) Expression in the Hippocampus.
AhR is a ligand-activated transcription factor [26] that modulates iNOS expression [27,28]. Terefore, we analyzed its expression in SAMP8 and SAMR1 mice. NYT1000 tended to decrease AhR expression in the hippocampus, although AhR levels were similar in SAMP8 and SAMR1 mice (Figure 6(a)). Tere were no diferences in frontal cortical AhR levels between SAMP8 and SAMR1 mice (Figure 6(b)). Te efect of NYT on AhR expression appeared to parallel its efect on iNOS expression. Terefore, AhR may be involved in the NYTinduced reduction in iNOS levels in the hippocampus.

SAMP8 Mice Have Increased Soluble Guanylate Cyclase β1
Expression. Te nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signaling pathway is implicated in depression-like behavior [18,[29][30][31][32][33]. Terefore, we analyzed sGCβ1 expression in the mice. In the hippocampus, sGCβ1 expression was higher in SAMP8 mice than in SAMR1 mice (Figure 2(a)), suggesting that increased sGCβ1 levels are associated with depressionlike behavior. However, NYT did not afect sGCβ1 expression. Given that NO increases sGCβ1 activity, the NYT-induced downregulation of iNOS may decrease the activity, but not the expression of sGCβ1 by reducing NO production.

Discussion
Kampo-hozai, a group of Kampo medicines that includes NYT [34], can make up for defciencies in physical and mental energy. Recent clinical studies have shown that NYT ameliorates depressive symptoms in older patients [8][9][10]. In the present study, we examined the mechanisms underlying the antidepression-like efect of NYT using SAMP8 mice, which exhibit accelerated aging. Treatment with NYT from a young age prevented onset of depression-like behavior in these animals. An iNOS inhibitor also ameliorated the depression-like behavior. Furthermore, NYT decreased iNOS expression in the hippocampus of SAMP8 mice. Tese fndings suggest that NYT exerts a prophylactic efect on depression-like behavior by decreasing iNOS levels.
Herbal extracts or components, which are included in NYT, are reported to afect iNOS expression. Panax ginseng extract has an anti-depressive efect and suppresses iNOS expression in a mouse model of stress-induced depression [35]. Catalpol, a component of Rehmanniae radix, also inhibits hippocampal iNOS gene expression and alleviates depression-like behavior in animal models of depression induced by chronic mild stress or chronic corticosterone administration [36,37]. Atractylenolide III, a component of Atractylodis rhizoma, has an antidepression-like efect in rats [38] and attenuates iNOS expression in lipopolysaccharide (LPS)-treated microglia [39]. Furthermore, Polygalae radix extract inhibits iNOS expression in LPS-treated microglia [40]. Extract of Astragali radix and components of Rehmanniae radix (2,5-dihydroxyacetophenone), Schisandrae fructus (Schisandrin), and Glycyrrhizae radix (total favonoids) also inhibit iNOS gene expression in a macrophage cell line [41][42][43][44]. Given that these components and extracts inhibit iNOS expression in microglia and macrophages, NYT may also suppress iNOS expression in these cells.
AhR is widely expressed in the brain, including the hippocampus [50,51]. Neurons, microglia, astrocytes, and oligodendrocytes all express AhR [52]. AhR modulates depression-like behavior, and AhR antagonism ameliorates depression-like behavior induced by LPS [53]. Furthermore, AhR KO mice show attenuation of depression-like behavior [54]. Terefore, NYT may attenuate depression-like behavior in SAMP8 mice in part by reducing AhR expression. Indeed, AhR KO mice show inhibition of the LPS-induced increase in iNOS expression [28]. Given these observations, NYT may reduce iNOS expression by inhibiting AhR signaling, thereby ameliorating depression-like behavior.
Te present study has at least two limitations. First, iNOS expression did not difer between SAMR1 and SAMP8 mice. Terefore, we did not demonstrate direct association between iNOS expression and depression-like behavior. However, administration of iNOS inhibitor to SAMP8 mice induced antidepression-like efects. Te iNOS inhibitor also decreased the immobility time of normal Swiss mice in a forced swimming test, which is another test that examines the efects of antidepressants [55]. Tese results suggest that iNOS inhibition indirectly afects the neuropathology of depression-like behavior in SAMP8 mice. Second, we could not measure cGMP or NO levels. Te NO/sGC/cGMP signaling pathway is implicated in depression-like behavior [18,[29][30][31][32][33]. Terefore, measuring these levels would help clarify whether NYT ameliorates depression-like behavior in mice with accelerated senescence by modulating NO and cGMP levels. It has been reported that NO levels are increased in the SAMP8 mouse brain [56,57], while cGMP levels in the brain have not been investigated to our knowledge. sGC is a key enzyme in the NO signaling pathway. Te binding of NO to sGC leads to a signifcant increase in cGMP [58]. Te present study demonstrated an increase in sGCβ1 levels in the hippocampus of SAMP8 mice, implying that cGMP levels may be increased in SAMP8 mice. Given that iNOS expression was not increased in SAMP8 mice, increased sGCβ1 levels may trigger depression-like behavior of SAMP8 mice.

Conclusions
Accelerated aging is associated with depression-like behavior in mice. Chronic treatment of NYT prevented onset of depression-like behavior and decreased hippocampal iNOS expression. Our fndings suggest that NYT may alleviate the onset and development of late-life depression in humans by downregulating iNOS expression in the brain.

Data Availability
Te datasets generated for this study are available from the corresponding author upon request.

Ethical Approval
Tis animal study was reviewed and approved by the Experimental Animal Care and Use Committee of Fukuoka University (#1714148 and #1915122).

Conflicts of Interest
Katsunori Iwasaki received a research grant from Tsumura & Co.

Authors' Contributions
CT performed the experiments, analyzed the data, and wrote the manuscript. TW designed the study, performed the experiments, analyzed the data, and wrote the manuscript. MH and AH validated the results and reviewed the manuscript. KK, SK, and KI reviewed and edited the manuscript.

Supplementary Materials
Supplementary  Evidence-Based Complementary and Alternative Medicine 7